Prodrug and profluorescent compounds for selective mitochondrial imaging and therapeutic targeting

ABSTRACT

The present invention relates to the use of prodrugs susceptible to nitroreductase (NTR) activation. In particular, provided herein are mitochondria-targeting prodrug compounds and probes, including profluore scent near-infrared (NIR) probes and non-fluorescent prodrugs, as well as to methods of using said prodrug compounds and probes for imaging mitochondria and for mitochondria-specific delivery of therapeutic agents.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication No. 62/350,557, filed Jun. 15, 2016, which is incorporatedherein by reference as if set forth in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not applicable.

BACKGROUND

Mitochondria are intracellular organelles responsible for a number ofmetabolic transformations and regulatory functions. They produce most ofthe ATP employed by eukaryotic cells. They are also the major source offree radicals and reactive oxygen species (ROS) that cause oxidativestress. Recent publications have the described the potential importanceof mitochondrial targeting for the development of new therapeutic agentssuch as anticancer agents.¹ For example, the selective induction ofelevated ROS levels in mitochondria has been found to produce differenteffects in cancer cells than in normal cells.¹ Several types of agentswhich affect mitochondrial function have been the focus of preclinicalstudies,² which argues for the relevance of this strategy. Whilemitochondrial localization has been reported for numerous molecules,³notably those containing a lipophilic cation,⁴ incorporating amitochondrial targeting moiety within a molecule may prove challengingin the broader context of molecular design for a specific cellulartarget.

Therefore, there remains a need in the art for improved tools forimaging mitochondria and for specifically targeting therapeutic agentsto mitochondria.

BRIEF SUMMARY

The present invention relates to the use of prodrugs susceptible tonitroreductase (NTR) activation. In particular, provided herein aremitochondria-targeting prodrug compounds and probes, includingprofluorescent near-infrared (NIR) probes, as well as to methods ofusing said prodrug compounds and probes for imaging mitochondria and formitochondria-specific delivery of therapeutic agents.

In a first aspect, provided herein is a prodrug compound having aformula selected from the group consisting of:

wherein X is selected from the group consisting of an alkylammoniumgroup (NR₃ ⁺), PR₃ ⁺, H, and an alkyl group, wherein Y is selected fromthe group consisting of O, NR, and S; wherein R is selected from thegroup consisting of H, an alkyl group, and an aromatic group; andwherein Drug represents any active drug having an alkylable heteroatom.

In another aspect, provided herein is a prodrug compound having aformula selected from the group consisting of:

wherein Z is an alkyl or styryl group, wherein A is H, SO₃H, or SO₃K;and wherein X is NR₃ ⁺, PR₃ ⁺, H, or an alkyl group.

A profluorescent compound having a formula selected from the groupconsisting of:

wherein X is selected from the group consisting of an alkylammoniumgroup (NR₃ ⁺), PR₃ ⁺, H, and an Alkyl group, wherein Y is selected fromthe group consisting of O, NR, and S; wherein R is selected from thegroup consisting of H, an alkyl group, and an aromatic group; andwherein Fluo represents any fluorescent compound having an alkylatableheteroatom.

In a further aspect, provided herein is a profluorescent compound havingthe formula, or an ortho-substituted analogue thereof:

wherein R₁ and R₃ are independently H, aryl or alkyl groups, and R₂ isan acyl group or a group chemically modifiable to an acyl group.

In yet another aspect, provided herein is a pharmaceutical compositioncomprising a prodrug or profluorescent compound as described herein anda pharmaceutically acceptable carrier.

Also provided herein is a method for imaging mitrochondria, where themethod comprises contacting the compound of any one of claims 1-4 to atarget cell; and detecting a nitrobenzyl fluorescence signal in one ormore mitochondria of the cell.

In another aspect, provided herein is a method for selective delivery ofa therapeutic agent to a mitochondrion of target cell, the methodcomprising contacting a target cell to a prodrug or profluorescentcompound operably linked to a therapeutic agent, whereby the therapeuticagent is selectively activated upon entry to a mitochondrion of targetcell.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will be better understood and features, aspects,and advantages other than those set forth above will become apparentwhen consideration is given to the following detailed descriptionthereof. Such detailed description makes reference to the followingdrawings, wherein:

FIG. 1 presents representative strategies for the release of active drugdepending of the use of a direct alkylation incorporating anortho-nitrobenyl or para-nitrobenzyl motif or through the introductionof self immolative linkers.

FIG. 2 presents chemical structures of exemplary prodrug forms ofmitochondria-targeting agents. Any drug exhibiting an alkylableheteroatom is suitable for design and use as a prodrug as provided inthis document.

FIG. 3 representative strategies for the release of active antioxidantdepending of the use of a direct alkylation incorporating anortho-nitrobenyl or para-nitrobenzyl motif or through the introductionof self immolative linkers.

FIG. 4 presents chemical structures of exemplary prodrug forms ofmitochondria-targeting antioxidants. Any antioxidant exhibiting analkylable heteroatom is suitable for design and use as a prodrug asprovided in this document.

FIGS. 5A-5B set forth exemplary mechanisms for nitroreductase (NTR)activation of fluorophore 3. (a) General mechanisms proposed for type Iand type II nitroreductases. (b) Mechanism of type I nitroreductasereduction applied to the activation of probe 2c.

FIGS. 6A-6D. (a) Selective activation of 2c in vitro by E. colinitroreductase. (b) Time dependent fluorescence emission by 2a, 2b and2c (excitation at 572 nm; emission at 703 nm) in PBS at 25° C. followingtreatment with E. coli NTR (1 μg mL⁻¹) and 0.5 mM NADH. (c) Absorbancespectrum evolution with time during incubation of probe 2c in 25 μM PBSbuffer, pH 7.4, with E. coli NTR (1 μg mL⁻¹) and 0.5 mM NADH. (d)Development of fluorescence from 10 μM 2c following treatment with 1 μgmL⁻¹ DT diaphorase (DTD) or nitroreductase (NTR) in 10 mM PBS buffer, pH7.4, for 20 min at 25° C. with or without 50 mM NADH.

FIGS. 7A-7D demonstrate fluorescence emission from 2c colocalized tomitochondria in live A549 cells. Cells were stained with (a) 2.5 μg mL⁻¹DAPI for nuclear staining, (b) 100 nM MitoTracker Green FM formitochondrial staining, (c) 10 μM 2c for NTR detection, and (d) overlayof a, b and c.

FIGS. 8A-8C demonstrate use of NTR for antimycin A (AMA) release inmitochondria. (a) Syntheses of p-nitrobenzyl-O-AMA (4a) and benzyl-O-AMA(4b) through alkylation of AMA. (b) Time course of appearance of AMAfluorescence (λ_(em)˜425 nm)²⁰ during microscopy experiments in liveA549 cancer cells treated with AMA and its alkylated derivatives 4a and4b at 25 μM concentrations. The need for high concentrations of 4a/4breflected the low quantum yield of the released AMA (˜0.06). (c) AMAfluorescence signal after release by nitroreductase from alkylatedanalogues of AMA. Free AMA appeared exclusively in the mitochondriafollowing treatment with compound 4a, while treatment with 4b resultedin no significant release of AMA.

FIGS. 9A-9B present biological activity of AMA, 4a and 4b (a) NADHoxidase activity assays on AMA, 4a and 4b at 0.05, 0.1 and 0.5 μMconcentrations. (b) Toxicity of AMA 4a and 4b toward A549 lung cancercells after 24 h of incubation at 1, 5 and 10 μM concentrations.

FIG. 10 illustrates exemplary synthesis protocols for NTR activatableprofluorescent probes 2a-c.

FIG. 11 is a fluorescence emission time course of 2c at differentconcentrations in PBS at 25° C. with NADH (0.5 mM) and E. colinitroreductase (1 μg mL⁻¹). Excitation was carried out at 572 nm and theemission was monitored at 703 nm.

FIG. 12 is a fluorescence emission spectrum (excitation at 572 nm) aftercomplete activation of 2c at different concentrations in PBS at 25° C.with 0.5 mM NADH and E. coli NTR (1 μg mL⁻¹). There was a linearcorrelation between fluorescence intensity and probe concentration.

FIG. 13 is a comparison of absorbance and emission spectra recorded inphosphate buffered saline (PBS), pH 7.4, at 25° C. of compound 3 andprobe 2c, after the later was activated with E. coli nitroreductase.

FIGS. 14A-14D present images of unactivated 2a in live A549 cells. Livecells were stained with (a) 2.5 μg mL⁻¹ DAPI for nucleus staining, (b)100 nM MitoTracker Green FM for mitochondria staining, (c) 10 μM 2a forNTR detection and (d) overlay of panels a, b, and c.

FIGS. 15A-15D present images of unactivated live A549 cells. Live cellswere stained with (a) 2.5 μg mL⁻¹ DAPI for nucleus staining, (b) 100 nMMitoTracker Green FM for mitochondria staining, (c) 10 μM 2b for NTRdetection and (d) overlay of panels a, b, and c.

FIG. 16 demonstrates activation of 2c by mitochondrial nitroreductase inlive A459 cells. (top) Visualization with red channel at differentconcentrations after 4 h incubation using an exposure time of 4.8 sec.(bottom) Quantitative analysis of the red fluorescent signal in A549cell mitochondria produced from 2c at different times and concentrationswith an exposure time of 4.8 sec.

FIG. 17 is a comparison of the spatial distributions of mitochondria andAMA in A549 cells treated with AMA.

FIG. 18 presents chemical structures of mitochondria-targetinganticancer agents useful for prodrug compounds according to theinvention.

DETAILED DESCRIPTION

All publications, including but not limited to patents and patentapplications, cited in this specification are herein incorporated byreference as though set forth in their entirety in the presentapplication.

The compounds, compositions, and methods provided herein are based atleast in part on the present inventors' discovery of nitroreductaseactivity in mitochondria. It was further discovered that nitroreductaseconverts novel prodrugs of the mitochondrial poison, antimycin A, into afluorescent, detectable compound upon entry into a mitochondrion.Without limitation, the present invention exploits the discovery that,upon, delivery of the prodrug to mitochondria of a target cell, it isconverted to AMA by the mitochondrial nitroreductase and exhibitsincreased cytotoxicity relative to AMA administered alone. Accordingly,provided herein are prodrugs susceptible to nitroreductase (NTR)activation, and compounds and methods for specific mitochondrial imagingand for selective delivery of therapeutic agents. In particular,provided herein are mitochondria-targeting prodrug compounds and probes,including profluorescent near-infrared (NIR) probes, as well as tomethods of using said prodrug compounds and probes for imagingmitochondria and for mitochondria-specific delivery of therapeuticagents.

I. Compounds and Compositions

Accordingly, in a first aspect, provided herein is a prodrug form of amitochondria-targeting compound, including compounds known to impactmitochondrial function. Preferably, the compound is susceptible tonitroreductase (NTR) activation in mitochondria. As used herein, theterm “prodrug” refers to compounds that are transformed (typicallyrapidly) in vivo to yield the parent compound of the above formulae, forexample, by enzymatic conversion in mitochondria. Synthesis of prodrugforms of such mitochondria-targeting agents will generally involvemodifying a heteroatom (usually oxygen) by alkylation with a nitrobenzylgroup. As used herein, the term “mitochondria-targeting” refers toagents that modulate mitochondrial activity in vivo or in vitro, andincludes drugs or chemical that be specifically delivered intomitochondria using, for example, conjugation to lipophilic cations orliposomes.

In exemplary embodiments, compounds of the invention are prodrugsreleasable with nitroreductase. Without being bound to any particularmechanism or theory, delivery of such prodrugs into mitochondria permitsmitochondria-specific release and activity of their active drug forms.As shown in FIG. 1, strategies for synthesizing prodrug forms of activedrugs involve a direct alkylation incorporating a ortho-nitrobenyl orpara-nitrobenzyl motif or the introduction of self immolative linkers.Chemical structures of exemplary prodrug forms of putativemitochondria-targeting agents as set forth in FIG. 2. Any drugexhibiting an alkylatable heteroatom is suitable for design and use as aprodrug as provided in this document.

In some cases, compounds of the invention are prodrug forms ofmitochondria-targeting anticancer agents. Several classes ofmitochondrial targeted anticancer agents, known as ‘mitocans’, have beenreported and categorized into eight classes depending on their sites ofaction, including (1) hexokinase inhibitors; (2) Bcl-2 family proteinligands; (3) thiol redox system disruptors; (4) mitochondrial membranetransporter/channel inhibitors; (5) electron transfer chainderegulators; (6) inner mitochondrial membrane disruptors; (7) TCA cycleinhibitors; and (8) mtDNA damaging agents. See, for review and exemplarychemical formulas of mitochans, Ma et al., Bioorganic & MedicinalChemistry Letters 25 (2015) 4828-4833. Exemplary mitocans are presentedin FIG. 18.

In some cases, compounds of the invention are prodrug forms ofantioxidants including mitochondria-targeting antioxidants. For example,referring to FIG. 3, prodrug forms of antioxidants can be synthesizedusing direct alkylation for incorporation of an ortho-nitrobenyl orpara-nitrobenzyl motif, or through the introduction of self-immolativelinkers. Exemplary prodrug mitochondria-targeting antioxidants include,without limitation, compounds having the structures shown in FIG. 4. Asdescribed herein, any drug exhibiting an alkylable heteroatom issuitable for synthesis and use as a prodrug as provided in thisdocument.

In a preferred embodiment, provided herein is a prodrug form ofantimycin A (AMA). The prodrug form of AMA is a profluorescent probe,meaning that the compound serves as a molecular probe comprising a cagedfluorochrome. As used herein, the term “caged fluorochrome” refers to anonfluorescent chemical compound that becomes fluorescent uponactivation or unmasking. While AMA is fluorescent, the profluorescentprobes described herein are not fluorescent in the absence ofmitochondrial nitroreductase activity. Without being bound to anyparticular mechanism or mode of action, it is believed thatmitochondrial nitroreductase activity catalyzes the conversion of theprodrug to AMA and “unmasks” a detectable fluorescent probe by enzymaticreduction of a nitro group. It will be appreciated, however, that thereleased drug need not be fluorescent to exert its therapeutic effects.Compounds of the invention also encompass prodrug forms ofnon-fluorescent compounds.

In some cases, the profluorescent probes provided herein areprofluorescent near infrared (NIR) compounds that are alkylatedderivatives of AMA. In some cases, the profluorescent probe has thefollowing chemical formula:

For Formula 4a, R₁ and R₃ are independently H, aryl or alkyl groups,including, including short linear and branched alkyl groups having 1 to10 carbon atoms (with substitutions). As used herein, an “alkyl group”means a linear, branched, or cyclic saturated hydrocarbon. Preferably,an alkyl group has between one and six carbon atoms. An alkoxy groupalso refers to substituted alkyl groups, which may include substituentssuch as alkanoyloxy groups, alkenyl groups, alkoxy groups, alkylsilylgroups, alkylsulfonyl groups, alkylsulfoxy groups, alkylthio groups,alkynyl groups, amino groups such as mono- and di-alkylamino groups andmono- and di-arylamino groups, amide groups, aryl groups, arylalkylgroups, carboxy groups, carboxyalkoxy groups, carboxyamide groups,carboxylate groups, haloalkyl groups, halogens, hydroxyl groups, nitrilegroups, nitro groups, phosphate groups, siloxy groups, sulfate groups,sulfonamide groups, sulfonyloxy groups, and combinations of these.Preferred substituents are alkoxy groups, amino groups such asdialkylamino groups, diarylamino groups, carboxylic acid-containinggroups, haloalkyl groups, halogens, hydroxyl groups, nitrile groups,nitro groups, and sulfonic acid groups. Examples of preferred alkylgroups include, but are not limited to, methyl, ethyl, propyl,isopropyl, cyclopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl,cyclobutyl, pentyl, isopentyl, neo-pentyl, 1-ethylpropyl, cyclopentyl,hexyl, and cyclohexyl. As used herein, a “halogen” means fluorine,chlorine, bromine, and/or iodide.

For Formula 4a, R₂ is an acyl group (RCO—, where R represents an alkylgroup that is linked to the carbon atom of the group by a single bond)such as a formyl, acetyl, propionyl, benzoyl, or acrylyl group. R₂ alsocan be a group that is modifiable to any acyl group. As used herein, an“acyl group” means a linear, branched, or cyclic substituent having acarbonyl group which is attached to either an oxygen atom, e.g., of ahydroxyl group, or a nitrogen atom, e.g., of an amino group. An acylgroup can include an alkoxy group, an alkyl group, an aryl group, anarylalkyl group, an ester group, an ether group a heterocyclic group, avinyl group, and combinations thereof. An acyl group also may besubstituted with substituents such as alkanoyloxy groups, alkenylgroups, alkylsilyl groups, alkysulfonyl groups, alkylsulfoxy groups,alkylthio groups, alkynyl groups, amino groups such as mono- anddi-alkylamino groups and mono- and di-arylamino groups, amide groups,carboxy groups, carboxyalkoxy groups, carboxyamide groups, carboxylategroups, haloalkyl groups, halogens, hydroxyl groups, nitrile groups,nitro groups, phosphate groups, siloxy groups, sulfate groups,sulfonamide groups, sulfonyloxy groups, and combination of these. Itshould be understood that an acyl group also can be an amino protectinggroup or a hydroxyl protecting group. As a hydroxyl protecting group, anacyl group may form an ester or carbonate. As an amino protecting group,an acyl group may form an amide or a carbamate. Examples of acyl groupsinclude, but are not limited to, alkoyl groups, aroyl groups, arylalkoylgroups, vinoyl groups. Preferred acyl groups are benzoyl, ethanoyl,tigloyl, or 2-methyl-2-butenoyl, 2-methyl-1-propenoyl, hexanoyl, butyrl,2-methybutyryl, phenylacetyl, propanoyl, furoyl, andtert-butyloxycarbonyl.

In some cases, the compound is an ortho-substituted analogue of Formula4a.

In another aspect, provided herein are compounds that are profluorescentforms of dyes for use in the near-infrared (NIR) wavelengths between 700and 900 nm. Such compounds are useful for nitroreductase detection inmitochondria. Furthermore, molecular probes that emit light in the NIRregion are particularly suited for in vivo and in vitro imaging. Asprofluorescent forms, the compounds provided herein are useful forspecific targeting of biomolecules of interest and reporting ofmolecular processes through fluorescence activation. General structuresof profluorescent probes for nitroreductase detection in mitochondriainclude the following:

where X=NR₃ ⁺, PR₃ ⁺H, Alkyl;

Y=O, NR, S; and

R=H, Alkyl, Aromatic.

The four schemes represent four strategies for the elimination of activefluorescent dye depending of the use of a direct alkylationincorporating a ortho-nitrobenyl or para-nitrobenzyl motif, or throughthe introduction of self-immolative linkers.

Compounds of the invention also include profluorescent forms of dyes foruse in the entire range of the UV/visible light spectrum such as, forexample, coumarins (and hybrids and derivatives thereof), xanthenefluorophores (e.g., rhodamin and fluoresceins), BODIPY, cyanine dyes,NIR fluorescent cyanine dyes, squaraine, and tetrapyrrole-basedcompounds, such as hematoporphyrin (HpD),meso-tetra-m-hydroxyphenylchlorin (m-THPC), benzoporphyrin derivatives(BPD), and sulfonated phthalocyanines. See, e.g., Zhang et al., CurrProtoc Cytom. 2012 April; Chapter 12:Unit 12.27. Synthesis of prodrug,profluorescent forms of such near-infrared dyes will generally involvemodifying a heteroatom (usually oxygen) by alkylation with a nitrobenzylgroup. In some cases, compounds are profluorescent forms of reactiveoxygen species (ROS)-activated near-infrared fluorescent dyes. Exemplarycompounds derived from known fluorophores structure include thefollowing:

where: Z=Alkyl, Styryl;

A=H, SO₃H, SO₃K; and

X=NR₃ ⁺, PR₃ ⁺, H, Alkyl.

It is to be noted that the styryl group may be either a styryl group nothaving a substituent, or a styryl group having a substituent. Likewise,the alkyl group may be either an alkyl group not having a substituent,or an alkyl group having a substituent.

The compounds of the invention are synthesized by an appropriatecombination of generally well-known synthetic methods. Techniques usefulin synthesizing the compounds of the invention are both readily apparentand accessible to those of skill in the relevant art. Exemplarysynthetic schemes leading to a probe of the invention is set forth inthe scheme in FIG. 10 and described in the Examples section below. Theseexamples illustrate certain of the diverse methods available for use inassembling the compounds of the invention, it is not intended to limitthe scope of reactions or reaction sequences that are useful inpreparing the compounds of the present invention.

Also provided herein are pharmaceutically acceptable salts ofprofluorescent probes. As used herein, the term “pharmaceuticallyacceptable salt” refers to derivatives of the compounds describedherein, wherein the parent compound is modified by making acid or basesalts thereof. Example of pharmaceutically acceptable salts include butare not limited to mineral or organic acid salts of basic residues suchas amines; and alkali or organic salts of acidic residues such ascarboxylic acids. The pharmaceutically acceptable salts include theconventional non-toxic salts or the quaternary ammonium salts of theparent compound formed, for example, from non-toxic inorganic or organicacids. Such conventional non-toxic salts include those derived frominorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic,phosphoric, and nitric acids; and the salts prepared from organic acidssuch as acetic, propionic, succinic, glycolic, stearic, lactic, malic,tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic,glutamic, benzoic, salicylic, sulfanilic, 2-acetoxybenzoic, fumaric,toluenesulfonic, naphthalenesulfonic, methanesulfonic, ethanedisulfonic, oxalic, and isethionic salts.

The pharmaceutically acceptable salts of the compounds can besynthesized from the parent compound, which contains a basic or acidicmoiety, by conventional chemical methods. Generally, such salts can beprepared by reacting the free acid or base forms of these compounds witha stoichiometric amount of the appropriate base or acid in water or inan organic solvent, or in a mixture of the two; generally, non-aqueousmedia like ether, ethyl acetate, ethanol, isopropanol, or acetonitrileare preferred. Lists of suitable salts are found in Remington'sPharmaceutical Sciences, 20th ed., Lippincott Williams & Wilkins,Baltimore, Md., 2000, p. 704; and “Handbook of Pharmaceutical Salts:Properties, Selection, and Use,” P. Heinrich Stahl and Camille G.Wermuth, Eds., Wiley-VCH, Weinheim, 2002.

Profluorescent compounds as described herein can be purified by any ofthe means known in the art, including chromatographic means, such asHPLC, preparative thin layer chromatography, flash column chromatographyand ion exchange chromatography. Any suitable stationary phase can beused, including normal and reversed phases as well as ionic resins. Mosttypically the disclosed compounds are purified via silica gel and/oralumina chromatography. See, e.g., Introduction to Modern LiquidChromatography, 2nd Edition, ed. L. R. Snyder and J. J. Kirkland, JohnWiley and Sons, 1979; and Thin Layer Chromatography, ed E. Stahl,Springer-Verlag, New York, 1969.

II. Methods of Use

The compounds of the invention are useful as probes, indicators, drugs,and the like. For example, a profluorescent compound can be used as adetectable marker of mitochondria for applications such as imaging oflive cells. In some cases, the method comprises contacting a target cellto a profluorescent compound and exposing the target cell to light at awavelength that is excitatory for a selected fluorochrome, and detectinga fluorescence emission, if present. Upon conversion, the profluorescentcompounds described herein exhibit an emission wavelength in the NIRregion, which affords good cell imaging efficiency by diminishing thebackground and enhancing light penetrability. The presence offluorescence emission at the expected wavelength is an indicator thatmitochondrial nitroreductase activity converted the profluorescentcompound to AMA. It may be noted further that non-fluorescent prodrugsof mitochondrially active compounds may be delivered selectively tomitochondria in their active form by the localized action of amitochondrial nitroreductase.

In some cases, the method comprises contacting a living target cell to aprofluorescent compound provided to the cell in a suitable culturemedium. For live cell imaging, the contacted cell can be analyzed on aninverted fluorescence microscope. Preferably, a heated stage is used tomaintain the cells at a suitable temperature. The suitable culturemedium and a suitable stage temperature will depend on the type of cellbeing viewed, and can be readily determined by one of ordinary skill inthe art. For long observation times involving cultured mammalian cells,it can be advantageous to chamber the microscope in 5% CO₂ to maintaincell viability. For example, as described in the Examples for compound4a, A549 cells (adenocarcinomic human alveolar basal epithelial cells)were cultured in RPMI-1640 medium at 37° C. in 5% CO₂, a usefulconcentration of 4a for imaging applications was 25 and the observationtime was 4 hours.

Digital imaging fluorescence microscopy is known in the art. Completedigital imaging fluorescence microscopy systems, or components forassembly of a complete system, are commercially available. Generally, abasic digital imaging fluorescence microscopy system includes thefollowing operationally linked components: (1) a conventionalfluorescence microscope, (2) a means for optical sectioning, e.g., amicrometer, (3) an optical detector, e.g., a CCD camera, and (4) acomputer or other storage medium to store optical data. The foregoingbasic components are commercially available. The operational linkage ofthe basic components is within ordinary skill in the art. Moreover,complete digital imaging fluorescence microscopy systems arecommercially available (e.g., Scanalytics, Billerica, Mass.).

III. Therapeutic Applications

The profluorescent compounds provided herein are useful for specifictargeting of therapeutic agents to mitochondria of a target cell.Accordingly, in another aspect, the present invention provides methodsfor treating a target cell or subject by administering a therapeuticallyeffective amount of a pharmaceutical composition comprising aprofluorescent compound and a pharmaceutically acceptable carrier. Asused herein, the term “treating” includes partially or completelyalleviates, ameliorates, relieves, inhibits, delays onset of, reducesseverity of and/or reduces incidence of one or more symptoms or featuresof a particular disease, disorder, and/or condition, and/or preventingor eliminating said symptoms. As used herein, the term “treating”indicates an improvement, increase, or reduction in values that arerelative to a baseline measurement, such as a measurement in the sameindividual prior to initiation of the treatment described herein, or ameasurement in a control individual (or multiple control individuals) inthe absence of the treatment described herein. Such treatment may be ofa subject who does not exhibit signs of the relevant disease, disorderand/or condition and/or of a subject who exhibits only early signs ofthe disease, disorder, and/or condition. Alternatively or additionally,such treatment may be of a subject who exhibits one or more establishedsigns of the relevant disease, disorder and/or condition.

“Subject” refers to a warm blooded animal such as a mammal, preferably ahuman, or a human child, which is afflicted with, or has the potentialto be afflicted with one or more diseases and disorders describedherein.

“Therapeutically effective amount” refers to that amount of a compoundwhich, when administered to a subject, is sufficient to effect treatmentfor a disease or disorder described herein. The amount of a compoundwhich constitutes a “therapeutically effective amount” will varydepending on the compound, the disorder and its severity, and the age ofthe subject to be treated, but can be determined routinely by one ofordinary skill in the art.

The methods can be used to treat or suppress conditions including butnot limited to cancer, obesity, atherosclerosis, amyotrophic lateralsclerosis, Parkinson's Disease, heart failure, myocardial infarction(MI), Alzheimer's Disease, Huntington's Disease, schizophrenia, bipolardisorder, fragile X syndrome, chronic fatigue syndrome, and Leighsyndrome, in a subject by administering an effective amount of acompound as described above including a salt or solvate or stereoisomerthereof.

For therapeutic methods, the profluorescent compounds provided hereinare preferably formulated as compositions comprising one or moreadditional active agents. For example, a profluorescent probe can beoperably linked to one or more active agents. As used herein, the term“operably linked” refers to a juxtaposition wherein the components areconfigured so as to perform their usual function. For example, amitochondria specific compound operably linked to therapeutic agent willdirect the linked agent to be localized to the mitochondria. Withoutbeing bound to any particular mechanism or mode of action, it isbelieved that mitochondrial nitroreductase activity catalyzes theconversion of the profluorescent compound to AMA and “unmasks” adetectable fluorescent probe and selectively releases or activates theactive agent for mitochondria-specific activity. The linked agentmaintains biological activity in the mitochondria. Suitable classes ofactive agents include, but are not limited to, antibiotic agents,antimicrobial agents, anti-acne agents, antibacterial agents, antifungalagents, antiviral agents, steroidal anti-inflammatory agents,nonsteroidal anti-inflammatory agents, anesthetic agents,antipruriginous agents, antiprotozoal agents, antioxidants,antihistamines, vitamins, and hormones.

In a particular embodiment, the therapeutic method is a method fortreating cancer in a subject in need thereof. The profluorescentcompounds described herein have increased toxicity toward cancer cellsrelative to antimycin A (AMA). In some cases, the method comprisingcontacting a target cell to a profluorescent compound described herein.The compound can be contacted to the target cell at a concentrationbetween about 0.5 μM and 20 μM (e.g., about 0.5, 0.75, 1, 1.5, 2, 2.5,5, 7.5, 10, 15, 20 μM). In exemplary embodiments, the method comprisingabout 1 μM to about 10 μM concentrations of a profluorescent compound.

In some cases, the therapeutic method is a method for treating adisorder or condition associated with a mitochondrial dysfunction.Suitable mitochondrial disorders that can be treated with thecompositions disclosed herein include but are not limited tomitochondrial myopathies. Mitochondrial myopathies include Kearns-Sayresyndrome, Leigh's syndrome, mitochondrial DNA depletion syndrome (MDS),mitochondrial encephalomyopathy, lactic acidosis and strokelike episodes(MELAS), myoclonus epilepsy with ragged red fibers (MERRF),mitochondrial neurogastrointestinal encephalomyopathy (MNGIE),neuropathy, ataxia and retinitis pigmentosa (NARP), and progressiveexternal ophthalmoplegia (PEO).

Mitochondria also represent an attractive target for cancer chemotherapydue to differences in mitochondrial utilization by normal and malignantcells. See, e.g., Glasauer and Chandel, Biochemical Pharmacology 92(2014) 90-101; Sullivan and Chandel, Cancer & Metabolism 2014, 2:17;Gorrini et al., Nat. Reviews 2013, 12:931; Jose et al., Biochimica etBiophysica Acta 1807 (2011) 552-561; Chandel, Cancer & Metabolism 2014,2:8; Fan et al., Molecular Systems Biology 2013, 9:712; and Wheaton etal., eLife 2014; 3:e02242. Since the delivery of anticancer agentsselectively to tumor cells represents a therapeutic challenge of longstanding, the selective activation of prodrugs of such compounds in themitochondria hold the potential for dramatically improved therapeuticoutcomes. Exemplary compounds for as mitochondria-targeting therapeuticagents as prodrugs include, without limitation, bleomycin (see, e.g.,Lim et al., Biochem. Pharmacol., 36, 2769-2774 (1987); Shen et al.,Mutat. Res., 337, 19-23 (1995); and Brar et al., Am. J. Physiol., 303,L413-L424 (2012)), metformin, procarbazine (Berneis et al., EuropeanJournal of Cancer 1966; 2:43-9), and the compounds set forth in FIG. 2.Other anti-cancer agents compounds include those that promote ROSproduction and, therefore, irreversible oxidative damage in tumor cellssuch as Arsenic trioxide (ATO), Elesclomol (STA-4783), rituximab, thepiperidine derivative lanperisone, and the ROS-producing azo derivativeof procarbazine. See, for review, Glasauer and Chandel, BiochemicalPharmacology 92 (2014) 90-101.

Formulations containing one or more of the compounds described hereinmay be prepared using a pharmaceutically acceptable carrier composed ofmaterials that are considered safe and effective and may be administeredto an individual without causing undesirable biological side effects orunwanted interactions. As generally used herein “pharmaceuticallyacceptable” refers to those compounds, materials, compositions, and/ordosage forms which are, within the scope of sound medical judgment,suitable for use in contact with the tissues of human beings and animalswithout excessive toxicity, irritation, allergic response, or otherproblems or complications commensurate with a reasonable benefit/riskratio. The carrier is all components present in the pharmaceuticalformulation other than the active ingredient or ingredients. Asgenerally used herein “carrier” includes, but is not limited to,diluents, binders, lubricants, disintegrators, fillers, pH modifyingagents, preservatives, antioxidants, solubility enhancers, and coatingcompositions.

Pharmaceutical compositions including the disclosed compounds areprovided. The pharmaceutical compositions may be for administration byoral, parenteral (intramuscular, intraperitoneal, intravenous (IV) orsubcutaneous injection), transdermal (either passively or usingiontophoresis or electroporation), transmucosal (nasal, vaginal, rectal,or sublingual) routes of administration or using bioerodible inserts andcan be formulated in dosage forms appropriate for each route ofadministration. In a preferred embodiment, the compounds areadministered orally. In another embodiment, the compounds areadministered parenterally in an aqueous solution.

The selected dosage depends upon the desired therapeutic effect, on theroute of administration, and on the duration of the treatment desired.Generally dosage levels of 0.001 to 10 mg/kg of body weight daily areadministered to mammals. Generally, for intravenous injection orinfusion, dosage may be lower.

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions illustrate the invention in a non-limiting fashion.

Example 1: Mitochondrial Nitroreductase Activity Enables SelectiveImaging and Therapeutic Targeting

Nitroreductase activities have been known for decades, studiedextensively in bacteria, and also in systems as diverse as yeast,trypanosomes and hypoxic tumors. The putative bacterial origin ofmitochondria prompted us to explore the possible existence ofnitroreductase activity within this organelle, and to probe its behaviorin a cellular context. Presently, by the use of a profluorescent nearinfrared (NIR) dye, we characterize the nature of a nitroreductaseactivity localized in mammalian cell mitochondria.

An alternative strategy might involve the organelle-specific enzymaticactivation of an inactive prodrug,⁵ e.g., by an enzymatic activityabundant within the organelle.⁶ The generally accepted bacterial originof mitochondria⁷ led us to investigate bacterial enzymatic activities ofpotential utility for mitochondrial prodrug activation. The presence ofredox enzymes in bacteria is now well established,⁸ and bacterialnitroreductase has been reported to reduce nitroaryl compounds to thecorresponding (hydroxy)anilines, apparently by one of two mechanisms.⁹One of these (type I) involves an initial two-electron reduction ofnitro groups and is tolerant of oxygen, permitting nitro group reductionto proceed to completion (FIG. 5a ). Type II nitroreductase, incontrast, involves an initial one-electron reduction and proceeds tocompletion only under hypoxic conditions.⁹ While nitroreductase activityin human cells appears to have been reported thus far only in hypoxictumors,¹⁰ early reports of nitroreductase activity in mitochondrialfractions isolated from mammalian liver cells¹¹ suggested the possibleexistence of a type I mitochondrial nitroreductase activity.

The detection of nitroreductase activities within hypoxic tumor tissueusing fluorogenic methods has been described recently, as reviewed byElmes,¹² and these presumably involve type II nitroreductase.Fluorescent probes have also been designed to measure small chemicalspecies within mitochiondria.^(4,13) In comparison, there appears tohave been no enzymatic activity detected in mitochondria using afluorogenic method.

In an effort to detect a mitochondrial nitroreductase which could beused for mitochondrial imaging, we designed a positively charged⁶fluorogenic probe which could potentially be triggered by anitroreductase.¹⁴ The QCy7 system¹⁵ seemed to be a good candidate. Itexhibits an emission wavelength in the NIR region which affords goodcell imaging efficiency by diminishing the background and enhancinglight penetrability. Also, the presence of indoles permitted theintroduction of positive charge by simple indole alkylation (FIG. 10).The preparation of ortho, meta and para substituted4-nitrobenzyloxy-isophthalaldehydes provided the alkylated precursors(1a-c) required for synthesis of the protected QCy7 dyes. Thedialdehydes were used for preparation of three new probes by treatmentwith an excess of 1-ethyl-2,3,3-trimethyl-3H-indolium iodide (pyridine,80° C., 3 h) as outlined in FIG. 10.

The strategy for the release of the fluorescent NIR dye from anon-fluorescent precursor via mitochondrial nitroreductase-mediatedreduction involved successive reduction, then elimination (FIG. 5b ).The use of ortho, meta and para substituted probes (2a-2c) was crucialbecause it enabled the investigation of the proposed transformation ingreater detail. Even if the reductive elimination of the ortho and parasubstituted probes proved to be practicable, none should occur in thecase of meta analogue 2b. To assess the viability of these probes forthe detection of nitroreductase activity, we first validated the probesin a cell free system using purified E. coli nitroreductase. Probes 2a-c(10 μM) were incubated in PBS buffer, pH 7.4, containing 0.5 mM NADH.After a short time, during which no fluorescence was observed, thenitroreductase was added (1 μg mL⁻¹) and the development of fluorescencewas monitored as a function of time (FIGS. 6b, 6c ). As expected, nofluorescence increase was observed for the probe 2b (metasubstituted).¹⁶ Conversely, good activation was observed for 2a and 2c,which are ortho and para-substituted, respectively. This confirmed whatwe expected, but also revealed that the activation of 2c was much fasterthan the activation of 2a, and increased with increasing substrateconcentration (FIGS. 11 and 12). This can be explained by a betterbinding of the para-nitrobenzyl compounds in the nitroreductase enzymeactive site as reported recently by Li et al.¹⁰ The selectivity of theprobe was also investigated using DT diaphorase, another two-electronreductase capable of activating small molecules.¹⁷ As shown in FIG. 6d ,significant activation occurred only in the presence of thenitroreductase+NADH. The monitoring of the absorbance spectrum duringthe NTR activation of 2c showed an increase of the absorbance bandcentered at 572 nm. This confirms that the released fluorescent dye iseffectively of the QCy7 type by comparison with an authentic standardprepared by chemical synthesis (FIG. 13). With the viability of thisprobe thus confirmed, we carried out microscopy experiments employingA549 cancer cells. The incubation of 10 μM 2c with the cells for 4 hourswas followed by the addition of 100 nm mitotracker green (40-minuteincubation). The results clearly show the appearance of a red signal(FIG. 7c ) characteristic of the release of QCy7 dye 3. The greenchannel (FIG. 7b ) illustrates visualization of the mitochondria bymitotracker green. Superposition of panels a-c (FIG. 7d ) shows completeoverlap of the red and green signals, resulting in a yellow signalaround the nucleus (stained in blue using DAPI).¹⁸ As a control, probes2a and 2b have also been tested, and neither provided a good signal(FIGS. 14 and 15). For 2b this is understandable as no eliminationprocess is possible, as confirmed during the in vitro tests. In the caseof 2a, we assume that the low kinetics of the reduction (c.f. FIG. 6b )is probably the cause of the absence of a strong signal. The appearanceof a signal from 2c was also concentration dependent (FIG. 16). Thisconstitutes the first direct visualization of nitroreductase activitylocalized in mitochondria. Notably, no hypoxic condition was required,i.e. the observed activity must be of type I, but this does not excludethe possible additional presence of type II nitoreductases in themitochondria.

The finding of nitroreductase activity associated with the mitochondriaof human cells encouraged us to design a new prodrug based on themitochondrial poison antimycin A (AMA). It has recently been noted thatAMA is toxic only at relatively high concentration,¹⁹ plausibly due to alack of facile access to the mitochondria. Further, it has been shownthat there is at least one locus of action for AMA which does notinvolve the respiratory chain.²⁰ By O-alkylating AMA with ap-nitrobenzyl moiety, it seemed possible that we might facilitatemitochondrial delivery and release the active form of the compound onlyin the mitochondria. Accordingly, O-p-nitrobenzyl-AMA (4a) was preparedin good yield by alkylation of AMA (FIG. 8a ). Also prepared as acontrol was O-benzyl-AMA (4b). Compounds 4a and 4b lack fluorescencewhile AMA is fluorescent.²⁰ In comparison, A549 cells treated with AMAresulted in a distribution of AMA which did not fully colocalize withmitochondria (FIG. 17). Accordingly, it is possible to monitor therelease of AMA within the mitochondria after enzymatic reduction of thenitro moiety in 4a. As anticipated, AMA was released from 4a by theaction of mitochondrial nitroreductase, but not to a significant extentfrom 4b (FIG. 8b ) and its appearance was localized to the mitochondria(FIG. 8c ).²¹

The evaluation of 4a and 4b in comparison with AMA was carried out bycomparing their effects both on the activity of the mitochondrialrespiratory chain and on cell viability. As shown in FIG. 9a , AMA was apotent inhibitor of NADH oxidase, which measures the activity ofmitochondrial respiratory complexes I, III, and IV. While AMA inhibitedNADH oxidase essentially completely at 50 nM concentration, 4a and 4bexhibited comparable, and much weaker activity. This is not surprisinggiven the putative importance of the phenolic moiety in AMA inassociation with the respiratory chain through hydrogen bonding.²² Inspite of their similar inhibitory activity toward NADH oxidase, 4a wasfound to be much more cytoxic toward A549 cancer cells than 4b, no doubtdue to the conversion of 4a to AMA within the mitochondria by thenitroreductase (FIG. 9b ). Compound 4a was considerably more cytotoxicthan exogenously added AMA, underscoring the belief that the latter isnot delivered efficiently to the mitochondria. This represents the firstexample of the use of a mitochondrial nitroreductase for selectivemitochondrial drug delivery, and should be extensible to numerous otherclasses of potential therapeutic agents.

In summary, this example demonstrates the identification of amitochondrial type I nitroreductase activity and the design andpreparation of a caged (non-fluorescent) probe. Our data demonstratesthat the caged probe is converted to a fluorescent compound by thenitroreductase when it enters cell mitochondria. By preparing a novelprodrug of the mitochondrial poison antimycin A, it is possible tofacilitate the delivery of antimycin to cell mitochondria, where it isunmasked by the mitochondrial nitroreductase and exhibits increasedcytotoxicity relative to antimycin A administered alone. This providesthe first example of an enzymatically activated mitochondrial probe.Further, we demonstrated that this enzymatic activity can be exploitedboth for the selective NIR imaging of mitochondria, and formitochondrial targeting by the activation of a mitochondrial poisonspecifically within that organelle. These findings represent the firstuse of a mitochondrial enzyme activity for unmasking agents formitochondrial fluorescent imaging and therapy, and may prove to be morebroadly applicable for imaging mitrochondria, and for selective deliveryof the active form of therapeutic agents designed to work within themitochondria of diseased cells.

Methods and Materials for Example 1

All experiments requiring anhydrous conditions were conducted inflame-dried glassware fitted with a rubber septum under a positivepressure of dry argon. Reactions were performed at room temperatureunless otherwise indicated. Analytical thin layer chromatography wasperformed using glass plates pre-coated with silica gel (0.25 mm, 60 Åpore size, 230-400 mesh, Silicycle) impregnated with a fluorescentindicator (254 nm). TLC plates were visualized by exposure toultraviolet light (UV) or by staining using I₂. Flash columnchromatography was performed employing silica gel (60 Å pore size, 40-63μm, standard grade, Silicycle).

Fluorescence quantum yields were measured at 25° C. by a relative methodusing cresyl violet (CV, Φ_(F)=56% in EtOH) as a standard.¹ Thefollowing equation was used to determine the relative fluorescencequantum yield:

Φ_(F)(x)=(A _(S) /A _(X))(F _(X) /F _(S))(n _(X) /n _(S))²Φ_(F)(s)

where A is the absorbance (in the range of 0.01-0.1 A.U.), F is the areaunder the emission curve, n is the refractive index of the solvents (at25° C.) used in measurements, and the subscripts s and x representstandard and unknown, respectively. The following refractive indexvalues were used: 1.362 for EtOH and 1.337 for PBS.

General Procedure for the Preparation of Nitrobenzylated4-Hydroxyisophthalaldehydes.

A solution containing 50.0 mg (0.33 mmol) of 4-hydroxyisophthalaldehydeand 91.2 mg (0.66 mmol) of K₂CO₃ in 2 mL of dry DMF was stirred underargon at room temperature for 10 min. The, 85.5 mg (0.40 mmol) ofnitrobenzyl bromide was added and the reaction mixture was stirred underargon overnight. After the reaction was completed (monitored by silicagel TLC using 2:1 hexanes-ethyl acetate), the reaction mixture wasdiluted with 50 mL of EtOAc and then washed with two 50-mL portions ofbrine and with 50 mL of water. The organic layer was dried (MgSO₄) andconcentrated under diminished pressure. The crude mixture wassolubilized in the minimum amount of CH₂Cl₂ and hexane was added toeffect the precipitation of a colorless solid. The precipitate wasisolated by filtration to give the desired compounds 1.

4-(o-Nitrobenzyloxy)isophthalaldehyde (1a)

The compound was prepared using 85.5 mg (0.40 mmol) of o-nitrobenzylbromide. Compound 1a was obtained as a colorless solid: yield 90.0 mg(96%); mp 145° C.; silica gel TLC R_(f) 0.60 (1:1 hexanes-ethylacetate); ¹H NMR (CDCl₃) δ 5.73 (s, 2H), 7.24 (d, 1H, J=8.7 Hz), 7.58(t, 1H, J=8.1 Hz), 7.77 (t, 1H, J=7.5 Hz), 7.92 (t, 1H, J=7.8 Hz), 8.14(m, 1H), 8.24 (d, 1H, J=8.2 Hz), 8.39 (m, 1H), 9.97 (d, 1H, J=0.7 Hz)and 10.58 (d, 1H, J=1.1 Hz); ¹³C NMR (CDCl₃) δ68.1, 113.7, 125.4, 125.5,128.5, 129.3, 130.5, 131.8, 132.7, 134.6, 136.2, 147.0, 164.1, 188.2 and190.0; mass spectrum (APCI), m/z 286.0711 (M+H)⁺ (C₁₅H₁₂N₂O₅ requiresm/z 286.0715).

4-(m-Nitrobenzyloxy)isophthalaldehyde (1b)

The compound was prepared using 85.5 mg (0.40 mmol) of m-nitrobenzylbromide. Compound 1b was obtained as a colorless solid: yield 88.3 mg(94%); mp 158° C.; silica gel TLC R_(f) 0.45 (1:1 hexanes-ethylacetate); ¹H NMR (CDCl₃) δ 5.40 (s, 2H), 7.20 (d, 1H, J=8.7 Hz), 7.65(dt, 1H, J=8.3 and 1.4 Hz), 7.83 (d, 1H, J=7.1 Hz), 8.14 (dt, 1H, J=8.6and 1.8 Hz), 8.26 (d, 1H, J=8.2 Hz), 8.34 (s, 1H), 8.38 (t, 1H, J=1.8Hz), 9.97 (d, 1H, J=1.4 Hz) and 10.55 (d, 1H, J=1.8 Hz); ¹³C NMR (CDCl₃)δ69.8, 113.4, 122.4, 123.8, 125.4, 130.3, 130.5, 132.4, 133.3, 135.9,137.2, 148.5, 164.2, 188.1 and 190.1; mass spectrum (APCI), m/z 286.0718(M+H)⁺ (C₁₅H₁₂N₂O₅ requires m/z 286.0715).

4-(p-Nitrobenzyloxy)isophthalaldehyde (1c)

The compound was prepared using 85.5 mg (0.40 mmol) of p-nitrobenzylbromide. Compound 1c was obtained as a colorless solid: yield 88.5 mg(94%); mp 185° C.; silica gel TLC R_(f) 0.45 (1:1 hexanes-ethylacetate); ¹H NMR (CDCl₃) δ 5.41 (s, 2H), 7.17 (d, 1H, J=8.7 Hz), 7.65(d, 2H, J=8.8 Hz), 8.12 (dd, 1H, J=8.7 and 2.2 Hz), 8.30 (d, 2H, J=8.8Hz), 8.38 (d, 1H, J=2.2 Hz), 9.97 (s, 1H) and 10.56 (s, 1H); ¹³C NMR(CDCl₃) δ69.8, 113.4, 124.3, 125.4, 127.9, 130.5, 132.5, 135.9, 142.2,148.2, 164.1, 188.1 and 190.0; mass spectrum (APCI), m/z 286.0714 (M+H)⁺(C₁₅H₁₂N₂O₅ requires m/z 286.0715).

4-(o-Nitrobenzyloxy)-QCy7 Probe (2a)

To a mixture of 20 mg (0.07 mmol) of4-(o-nitrobenzyloxy)-isophthalaldehyde, 48.5 mg (0.15 mmol) of1-ethyl-2,3,3-trimethyl-3H-indolium iodide and 12.6 mg (0.15 mmol) ofsodium acetate in 1 mL of dry acetic anhydride was applied a positivepressure of argon. The reaction mixture was stirred at 80° C. for 45minutes (min) under argon, leading to the precipitation of the desireddye as an orange solid. The solvent was removed by centrifugation andthe resulting solid was washed twice with acetic anhydride then twicewith Et₂O. The solid was dried under vacuum to furnish 2a as an orangesolid: yield 47 mg (76%); mp 178-180° C.; silica gel TLC R_(f) 0.30(95:5 CH₂Cl₂-MeOH); ¹H NMR (CDCl₃) δ 1.65 (m, 6H), 1.80 (s, 6H), 2.06(s, 6H), 5.11 (q, 2H, J=7.2 Hz), 5.18 (q, 2H, J=7.2 Hz), 5.79 (s, 2H),7.43 (d, 1H, J=9.0 Hz), 7.50-7.65 (m, 9H), 7.75 (m, 2H), 8.03-8.13 (m,2H), 8.29 (d, 1H, J=16.4 Hz), 8.64 (d, 1H, J=16.4 Hz), 9.04 (d, 1H,J=16.1 Hz), 9.36 (d, 1H, J=8.8 Hz) and 9.90 (s, 1H); ¹³C NMR (CDCl₃)δ14.8, 14.9, 21.2, 27.2, 44.3, 44.7, 52.7, 53.2, 69.0, 112.1, 113.9,114.2, 114.3, 114.6, 123.1, 123.2, 123.6, 125.5, 129.1, 129.4, 129.9,130.3, 130.4, 131.0, 134.2, 136.0, 139.6, 140.3, 144.0, 144.7, 148.2,148.5, 155.0, 161.8, 182.3 and 182.8; mass spectrum (APCI), m/z 624.3210(M−H)⁺ (C₄₁H₄₂N₃O₃ requires m/z 624.3226).

4-(m-Nitrobenzyloxy)-QCy7 Probe (2b)

To a mixture of 20 mg (0.07 mmol) of4-(m-nitrobenzyloxy)-isophthalaldehyde and 48.5 mg (0.15 mmol) of1-ethyl-2,3,3-trimethyl-3H-indolium iodide in 1 mL of dry pyridine wasapplied a positive pressure of argon. The reaction mixture was stirredat 80° C. for 5 hours (h) under argon. The cooled reaction mixture wascentrifugated to remove the solid residue. The supernatant was dilutedin 1 mL of CH₂Cl₂ and added dropwise to 20 mL of diethyl ether to inducethe precipitation of the desired compound. The resulting solid wasisolated by filtration, washed with Et₂O, dissolved in a minimum amountof CH₂Cl₂ and applied to a 1000μ preparative silica gel TLC plate (20×10cm) for purification. Elution with 97:3 CH₂Cl₂-MeOH afforded compound 2bas an orange solid: yield 17 mg (28%); mp 183-185° C.; silica gel TLCR_(f) 0.30 (95:5 CH₂Cl₂-MeOH); ¹H NMR (CDCl₃) δ (1.67 m, 6H), 1.83 (s,6H), 2.09 (s, 6H), 5.12 (q, 2H, J=7.3 Hz), 5.23 (q, 2H, J=7.3 Hz), 5.52(s, 2H), 7.52 (m, 1H), 7.54-7.60 (m, 5H), 7.60-7.64 (m, 3H), 7.69 (t,1H, J=8.1 Hz), 7.86 (d, 1H, J=7.7 Hz), 8.09 (d, 1H, J=16.2 Hz), 8.30 (d,1H, J=8.1 Hz), 8.39 (d, 1H, J=16.2 Hz), 8.51 (m, 1H), 8.83 (d, 1H,J=16.2 Hz); 9.15 (d, 1H, J=16.3 Hz), 9.59 (dd, 1H, J=9.1 and 1.9 Hz) and10.01 (d, 1H, J=1.9 Hz); ¹³C NMR (CDCl₃) δ14.9, 15.1, 27.2, 27.4, 44.2,44.9, 52.7, 53.3, 70.2, 112.1, 113.6, 114.1, 114.4, 114.6, 121.4, 123.2,123.7, 129.3, 129.5, 129.9, 130.3, 133.6, 136.1, 137.9, 140.0, 140.3,143.9, 144.8, 148.2, 148.7, 155.1, 161.8, 182.1 and 182.9; mass spectrum(APCI), m/z 624.3212 (M−H)⁺ (C₄₁H₄₂N₃O₃ requires m/z 624.3226).

4-(m-Nitrobenzyloxy)-QCy7 Probe (2c)

To a mixture 20 mg (0.07 mmol) of 4-(p-nitrobenzyloxy)-isophthalaldehydeand 48.5 mg (0.15 mmol) of 1-ethyl-2,3,3-trimethyl-3H-indolium iodide in1 mL of dry pyridine was applied a positive pressure of argon. Theresulting reaction mixture was stirred at 80° C. for 3 h under argonleading to the precipitation of the desired dye as an orange solid. Thesolvent was removed by centrifugation and the resulting solid was washedtwice with cold pyridine then twice with Et₂O. The solid was then driedunder vacuum to furnish compound 2b as an orange solid: yield 42.5 mg(65%); mp 203-205° C.; silica gel TLC R_(f) 0.30 (95:5 CH₂Cl₂-MeOH); ¹HNMR (CDCl₃) δ 1.66 (m, 6H), 1.77 (s, 6H), 2.07 (s, 6H), 5.12 (q, 2H,J=7.2 Hz), 5.19 (q, 2H, J=7.2 Hz), 5.50 (s, 2H), 7.5-7.65 (m, 9H), 7.74(d, 2H, J=8.6 Hz), 8.09 (d, 1H, J=16.3), 8.33 (d, 2H, J=8.6 Hz), 8.38(d, 1H, J=16.4 Hz), 8.74 (d, 1H, J=16.4 Hz), 9.09 (d, 1H, J=16.2 Hz),9.50 (dd, 1H, J=9.0 and 1.9 Hz) and 9.99 (d, 1H, J=1.9 Hz); ¹³C NMR(CDCl₃) δ14.9, 15.0, 27.2, 27.4, 44.3, 44.8, 52.5, 53.2, 70.6, 112.2,113.7, 114.2, 114.3, 114.6, 123.1, 123.2, 124.2, 128.6, 129.2, 129.5,129.9, 130.1, 130.4, 136.1, 140.1, 140.27, 140.29, 142.6, 143.7, 144.7,148.2, 148.5, 155.0, 161.9, 182.0 and 182.8; HRMS (APCI), m/z 624.3220(M−H)⁺ (C₄₁H₄₂N₃O₃ requires m/z 624.3226).

Acetyl-QCy7 (2d)

To a suspension containing 50.0 mg (0.33 mmol) of isophthalaldehyde, 218mg (0.69 mmol) of 1-ethyl-2,3,3-trimethyl-3H-indolium iodide and 83.9 mg(1.02 mmol) of sodium acetate in 5 mL of dry acetic anhydride wasapplied a positive pressure of argon. The resulting reaction mixture wasstirred at 80° C. for 45 min leading to the precipitation of the desireddye as an orange solid. The solvent was removed by centrifugation andthe resulting solid was washed twice with acetic anhydride and withEt₂O. The solid was then dried under vacuum to afford 2c as an orangesolid, which was used directly for the next step: yield 239 mg (92%); ¹HNMR (CDCl₃) δ (1.70 m, 6H), 1.87 (s, 6H), 2.09 (s, 6H), 2.47 (s, 3H),5.20 (q, 2H, J=7.4 Hz), 5.28 (q, 2H, J=7.4 Hz), 7.54-7.71 (m, 9H), 8.19(d, 1H, J=16.1 Hz), 8.32 (d, 1H, J=16.2 Hz), 8.44 (d, 1H, J=16.2 Hz),9.07 (d, 1H, J=16.3 Hz), 9.29 (d, 1H, J=8.7 Hz) and 10.00 (s, 1H); ¹³CNMR (CDCl₃) δ15.0, 15.2, 21.4, 27.0, 27.3, 44.8, 45.4, 52.9, 53.5,114.2, 114.6, 115.1, 115.5, 123.2, 123.3, 124.4, 126.8, 129.6, 130.1,130.3, 130.8, 133.3, 134.6, 137.6, 140.2, 140.3, 143.8, 144.9, 147.0,153.8, 154.1, 167.9, 182.0 and 183.0; mass spectrum (MALDI), m/z 266.46(M)²⁺/2 (theoretical m/z 266.15).

Diethyl QCy7 (3)

To a solution of 30.0 mg (38.1 μmol) of the crude solid 2d in 1 mL ofMeOH was added 5.25 mg (38.1 μmol) of K₂CO₃ and the resulting greensolution was stirred at room temperature for 4 h. The reaction mixturewas then diluted with 2 mL of CH₂Cl₂ and filtered through cotton toremove inorganic material. The filtrate was then concentrated underdiminished pressure the crude residue was dissolved in 3:1 CH₂Cl₂-AcOHand purified by flash chromatography on a silica gel column (15×1 cm).Elution with 95:2.5:2.5 CHCl₃-MeOH-AcOH afforded 3 as a yellow solid:yield 12 mg (51%); silica gel TLC R_(f) 0.25 (95:2.5:2.5CHCl₃-MeOH-AcOH); ¹H NMR (CDCl₃) δ 1.16 (m, 3H), 1.27 (s, 3H), 1.63 (t,3H, J=7.1 Hz), 1.79 (s, 6H), 2.09 (s, 3H), 3.21 (m, 1H), 3.32 (m, 1H),5.00-5.12 (m, 2H), 5.81 (d, 1H, J=10.2 Hz), 6.56 (d, 1H, J=7.7 Hz), 6.74(d, 1H, J=8.4 Hz), 6.83 (t, 1H, J=7.3 Hz), 7.07 (d, 1H, J=7.3 Hz), 7.17(t, 1H, J=7.7 Hz), 7.42 (t, 1H, J=7.2 Hz), 7.47-7.58 (m, 5H), 7.89 (d,1H, J=16.2 Hz), 8.06 (d, 1H, J=16.2 Hz) and 9.03 (s, 1H); ¹³C NMR(CDCl₃) δ14.3, 14.5, 26.3, 27.5, 28.2, 37.9, 44.1, 51.9, 52.8, 103.7,106.6, 109.7, 114.1, 116.1, 119.1, 121.0, 121.8, 122.7, 126.4, 126.6,127.8, 129.3, 129.4, 129.8, 130.1, 136.2, 136.4, 140.6, 143.1, 146.9,155.7, 155.7, 166.8 and 180.6; HRMS (APCI), m/z 489.2920 (M)⁺ (C₃₄H₃₇N₂Orequires m/z 489.2900). quantum yield 6.1% (PBS buffer, pH 7.4)

2-O-(p-Nitrobenzyl)AMA (4a)

A solution containing 10.0 mg (19.0 μmol) of antimycin A and 10.0 mg(76.0 μmol) of K₂CO₃ in 500 μL of dry CH₃CN was stirred under argon.After 15 min, 7.00 mg (38.0 μmol) of p-nitrobenzyl bromide and a crystalof NaI were added. The reaction mixture was stirred at room temperatureunder argon for 12 h. The reaction was monitored by silica gel TLC (2:1hexane-EtOAc) until it was complete. The crude reaction mixture wasdiluted with 1 mL of CH₂Cl₂ and filtered through cotton to removeinorganic material. The resulting solution was concentrated underdiminished pressure and the residue was purified by preparative silicagel TLC plate (1000μ, 20×10 cm) Elution with 1:1 hexane-EtOAc affordedcompound 4a as a colorless wax: yield 11 mg (84%); silica gel TLC R_(f)0.20 (1:1 hexanes-ethyl acetate); mass spectrum (APCI), AMA₁ m/z684.3142 (M+H)⁺ (C₃₅H₄₆N₃O₁₁ requires 684.3133); AMA₂ m/z 670.2965(M+H)⁺ (C₃₄H₄₄N₃O₁₁ requires 670.2976); AMA₃ m/z 656.2827 (M+H)⁺(C₃₃H₄₂N₃O₁₁ requires 656.2820); AMA₄ m/z 642.2659 (M+H)⁺ (C₃₂H₄₀N₃O₁₁requires 642.2663).

2-O-Benzyl-AMA (4b)

A solution containing 10.0 mg (19.0 μmol) of antimycin A and 10.0 mg(76.0 μmol) of K₂CO₃ in 500 μL of dry CH₃CN was stirred under argon.After 15 min, 4.60 μL (38.0 μmol) of benzyl bromide and a crystal of NaIwere added. The reaction mixture was stirred at room temperature underargon for 12 h. The reaction was monitored by silica gel TLC (2:1hexane-EtOAc) until the reaction was complete. The crude reactionmixture was diluted with 1 mL of CH₂Cl₂ and filtered through cotton toremove inorganic material. The resulting solution was concentrated underdiminished pressure and the residue was purified by preparative silicagel TLC plate (1000μ, 20×10 cm). Elution with 2:1 hexane-EtOAc affordedcompound 4b as a colorless wax: yield 11.2 mg (92%); silica gel TLCR_(f) 0.50 (1:1 hexanes-ethyl acetate); HRMS (APCI), AMA₁ m/z 639.3280(M+H)⁺ (C₃₄H₄₅N₂O₉ requires 639.3281); AMA₂ m/z 625.3141 (M+H)⁺(C₃₄H₄₅N₂O₉ requires 625.3125); AMA₃ m/z 611.2972 (M+H)⁺ (C₃₃H₄₃N₂O₉requires 611.2968); AMA₄ m/z 597.2813 (M+H)⁺ (C₃₂H₄₁N₂O₉ requires597.2812).

Fluorescence Monitoring.

A solution of fluorogenic probe was prepared in 1 mL of phosphatebuffered saline (PBS) and 150 μL of the resulting solution wastransferred into a quartz fluorescence cell. A volume of 1.51 μL of a 50mM solution of NADH was then added to afford a final concentration of0.5 mM and the resulting solution was incubated at 25° C. Afterexcitation at 572 nm the emission of the released fluorophore wasmonitored at 703 nm over time with measurements recorded every 1 sec.After 100 sec of incubation, 1.51 μL of a 1 mg mL⁻¹ solution of E. colinitroreductase in water was added in the quartz fluorescence cell andthe solution was mixed as the monitoring proceeded.

Absorbance Monitoring.

A solution of fluorogenic probe was prepared in 1 mL of phosphatebuffered saline (PBS) and 150 μL of the resulting solution transferredinto a quartz fluorescence cell. A volume of 1.51 μL of a 50 mM solutionof NADH was then added to afford a final concentration of 0.5 mM and theresulting solution was incubated at 25° C. After measuring theabsorbance spectrum of the probe at to, a volume of 1.51 μL of a 1mg·mL⁻¹ of E. coli nitroreductase in water was added in the quartzfluorescence cell and the solution was mixed. The absorption spectrum ofthe solution was measured between 400 and 700 nm every 2 min.

Fluorescence Microscopy Imaging

Activation of Probes 2a, 2b and 2c in Live A549 Cells.

Fluorescence images were acquired using a Zeiss Axiovert 200M invertedmicroscope fitted with an AxioCam MRm camera equipped with a 300W xenonlamp (Sutter, Novato, Calif.), and Texas red and FITC filters (Chroma,Bellows Falls, Vt.). Lung cancer A549 cells (ATCC CCL-185) were grown onglass bottom microwell disks (MatTek Corporation, MA, USA) at a celldensity of 10,000 cells/well at 37° C. for 24 hours. When the cellconfluence reached ˜60-70%, the cells were rinsed three times withphosphate buffered saline (PBS). The cells were treated with 2a, 2b, and2c at 1, 2.5, 5 and 10 μM concentrations for 1, 2, and 4 hours. Then thecells were stained using 2.5 μg/mL DAPI (Invitrogen) and 100 nMMitoTracker Green FM (Cell Signaling Technology, Inc.) for 40 min.Thereafter, all images were recorded and the target cells counted usinga 40× oil objective. To ensure accurate intensity measurements, theexposure time and laser time were kept the same. Pixel intensity wasquantified using AxioVision release 4.7 version software, and the meanpixel intensity was generated as gray level. Data are reported as themean of three independent experiments.

Activation of Prodrugs 4a and 4b in Live A549 Cells.

Fluorescence microscopy images were acquired using a Zeiss Axiovert 200Minverted microscope fitted with an AxioCam MRm camera equipped with a300W xenon lamp (Sutter, Novato, Calif., USA), and DAPI (blue channel)and Texas red (red channel) filters (Chroma, Bellows Falls, Vt., USA).A549 cells were grown on glass bottom microwell disks (MatTekCorporation, MA, USA) at a cell density of 10000 cells/well at 37° C.for 24 hours. When the cell confluence reached ˜60-70%, the cells wererinsed three times with phosphate buffered saline (PBS). The cells werethen treated with AMA, 4a and 4b at 25 μM concentration for 4 hours. Thecells were then stained using 100 nM MitoTracker Green FM (CellSignaling Technology, Danvers, Mass., USA) for 40 minutes. Thereafter,all images were recorded and counted using a 40× oil objective. Toensure accurate intensity measurements, the exposure time and laser timewere kept the same. Data are reported as the mean of three independentexperiments.

Biological Activity of AMA and Prodrugs 4a and 4b

MTT Toxicity Assays.

Exponentially growing A459 cells were harvested and plated in 96-wellplates at a concentration of 2×10⁴ cells/well. After incubation at 37°C. for 24 h, the cells were treated with compounds AMA, 4a and 4b atfinal concentrations of 1, 5 and 10 μM for an additional 24 hours. Then20 μL of MTT (5 mg/mL) was added to each well and the plates wereincubated at 37° C. for 4 h. The supernatants were discarded, and 100 μLof DMSO was added to each well. The absorbance was recorded at 490 nmafter 15 min. Cell viability was determined by the following formula:

cell viability (%)=100−(OD _(negative control) −OD_(treatment))×100%/(OD _(negative control) −OD _(background)).

Data are reported as the mean of three independent experiments, each runwas carried out quintuplicate.

NADH Oxidase Activity.

The effect of compounds AMA, 4a and 4b on NADH oxidase activity wereevaluated using bovine heart mitochondria. The bovine heart mitochondriaand bovine heart submitochondrial particles (SMPs) were prepared asdescribed.² The SMPs were diluted to 0.5 mg/mL, and the test compoundswere assayed at 25° C. and monitored spectrophotometrically using aBeckman Coulter DU-530 spectrometer (340 nm, ε 6.22 mM⁻¹ cm⁻). NADHoxidase activity was determined in 1 mL of 50 mM Hepes buffer containing5 mM MgCl₂, pH 7.5. The final mitochondrial protein concentration was 30μg/mL. The initial rates of NADH oxidation were calculated from thelinear portion of the traces. Data are reported as the mean of threeindependent experiments each run in triplicate.

REFERENCES AND FOOTNOTES

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1.-3. (canceled)
 4. A profluorescent compound having the formula, or anortho-substituted analogue thereof:

wherein R₁ and R₃ are independently H, aryl or alkyl groups, and R₂ isan acyl group or a group chemically modifiable to an acyl group. 5.-7.(canceled)